This invention relates to a high dynamic range apparatus for separation and detection of polynucleotide fragments which is particularly useful in performing the method for quantifying and partially sequencing a nucleic acid analyte that is present in a sample as disclosed in related U.S. patent application Ser. No. 08/819,912. In this method, a nucleic acid-containing sample is combined with a control polynucleotide and then amplified with multiple primer sets to produce control nucleic acid fragment generated by amplification of the control polynucleotide, conserved nucleic acid fragments generated by amplification of a conserved region of the nucleic acid in the sample to produce a quantitative measure of the presence of a target nucleic acid in the sample, and sequencing fragments preferably generated by amplification of a more variable portion of the nucleic acid in the sample which, upon sequencing, can provide information concerning and/or discrimination among different but closely related forms of a target nucleic acid, including but not limited to different allelic or mutant forms of genes, or in the case of microorganisms, subspecies, serovars, strains, sub-types, biovars, variants, or serotypes or between closely related species of the target. The resulting fragments are separated by size, for example by polyacrylamide gel electrophoresis (PAGE) and the control and conserved fragments are detected by fluorescence from a label attached to the amplification primer.
Depending on the differential amounts of control and target nucleic acid present in the sample, the magnitudes of the signals corresponding to the control and conserved fragments may differ by several orders of magnitude. In the past, because instruments used for measuring fluorescence from electrophoresis gels have had a relatively low dynamic range, this has generally meant that the replicate experiments at different dilutions must be performed to permit both a peaks to be measured. Such dilutions increase the cost of an assay, because of increased time required to prepare the sample and decreased instrumental throughput, and can be a source of error in the quantitation of the amount of material in the sample since serial dilutions necessitate both precise preparation and a post-measurement calculation to arrive at a quantitative result.
To maximize the ability to obtain quantitative results for the amount of a target nucleic acid in a sample, without having to perform serial dilutions, it would be desirable to have an apparatus for electrophoretic separation and real-time detection of nucleic acids which had a high dynamic range, for example on the order of 10.sup.4 to 10.sup.6 or more. It is an object of the present invention to provide such an apparatus.